The PHEX gene (formerly PEX; Phosphate regulating gene with homologies to Endopeptidases on the X chromosome) was identified by a positional cloning approach as the candidate gene for X-linked hypophosphatemia (XLH) (Francis et al., 1995). XLH is a Mendelian disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in phosphate re-absorption and vitamin D metabolism (Rasmussen and Tenenhouse, 1995). Using the information made available by the publication of the sequence of the PHEX gene, and standard techniques obvious to those skilled in the art, several groups have cloned and sequenced the human and mouse PHEX/Phex cDNAs (Du et al., 1996; Lipman et al., 1998; Grieff et al., 1997; Beck et al., 1997; Guo and Quarles, 1997; Strom et al., 1997) (PHEX/Phex refers to the human and mouse genes, respectively). Amino acid sequence comparisons have demonstrated homologies between PHEX/Phex protein and members of the neutral endopeptidase family as previously observed in the partial sequence of the candidate gene (Francis et al., 1995). The peptidases of the neutral endopeptidase family are zinc-containing type II integral membrane glycoproteins with a relatively short cytoplasmic N-terminal region, a single transmembrane domain, and a long extracytoplasmic domain, which contains the active site of the enzyme (Devault et al., 1987).
The mechanism by which loss of PHEX function elicits the bone and renal abnormalities observed in XLH patients is not clear. There are no data suggesting the presence of PHEX/Phex mRNA in the kidney (Du et al., 1996; Beck et al., 1997; Grieff et al., 1997). The increased renal phosphate excretion in Hyp mice is due to a down-regulation of the phosphate transporter, which is necessary for the re-absorption of the phosphate from the nephron (Tenenhouse 1998). The serum concentration of 1,25(OH)2D3 (calcitriol) was found to be the same in Hyp mice as in normal littermates (Meyer 1980). However, the Hyp kidney showed an accelerated degradation of the vitamin D metabolite to 1,24,25(OH)3D3, a metabolite with reduced activities (Tenenhouse 1988). In the presence of a phosphate rich diet, Hyp mice experienced an increase in serum 1,25(OH)2D3 and a fall in the C-24 oxidation products, while normal mice experienced no such changes (Tenenhouse 1990). To summarize, the renal disorder in vitamin D metabolism in Hyp mice appears to be secondary to the phosphate disorder.
PHEX/Phex mRNA was detected in bones by Northern blot hybridization and in other adult and fetal tissues such as lungs, liver, muscles, and ovaries by RT-PCR and RNase protection assays (Du et al., 1996; Beck et al., 1997). In situ hybridization performed on sections of embryos and newborn mice showed the presence of Phex mRNA in osteoblasts and odontoblasts (Ruchon et al., 1998). Phex gene expression was detectable on day 15 of embryonic development, which coincides with the beginning of intracellular matrix deposition in bones. Moreover, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the Phex transcript is decreased in adult bones and in non growing teeth. This result was confirmed when the presence of the Phex protein in newborn adult bones was investigated by Western blotting using a monoclonal antibody raised against the human PHEX. Immunohistochemical studies on a 2 month-old mouse showed exclusive labelling of mature osteoblasts and osteocytes in bones and of odontoblasts in teeth (Ruchon et al., 2000: J Bone Miner. Res. In press). Taken together these results suggest that PHEX/Phex plays an important role in the development and maintenance of mineralization in these tissues.
Further insights into the role of PHEX in bone metabolism were provided by experimental studies on cases of oncogenic osteomalacia (OOM), a tumor-associated sporadic condition with very similar clinical indications. There is strong evidence that a tumor-produced humoral factor inhibits renal phosphate re-absorption and vitamin D synthesis resulting in osteomalacia (Nelson et al., 1997). Experimental studies on Hyp and Gy mice, the murine model of human XL, also suggest the involvement of a humoral factor. In both mouse models, mutations have been identified in the Phex gene, which also appear to result in loss of function of the gene product (Strom et al., 1997; Beck et al., 1997).
Considering the similarities between PHEX protein and the other members of the metallopeptidase family of enzymes, it has been speculated that PHEX metabolizes a peptide hormone that modulates renal tubular phosphate re-absorption. Such an activity could involve either the processing of a phosphate reabsorbing hormone precursor to its active form or the inactivation of a circulating phosphaturic factor. There is evidence of intrinsic abnormalities in osteoblasts from Hyp mice (Ecarot et al., 1992). A defective phosphate transport was also observed in osteoblasts from Hyp mice (Rifas et al., 1994). PHEX might thus be involved in the control of bone metabolism both indirectly at the level of the kidney by controlling renal phosphate reabsorption and directly at the level of bones by inactivating a trophic peptide factor controlling either osteoblast or osteoclast functions or both.
Since absence of a functioning PHEX gene leads to hypophosphatemia, it should be possible to control human diseases involving hyperphosphatemia through the inhibition of this enzyme. Thus, inhibiting PHEX will cause a reduction in blood phosphate concentration, allowing for the prevention and reduction of hyperphosphatemiarelated disorders in humans and animals. Reduced renal excretion of phosphorus due to impaired kidney functions is the most common cause of hyperphosphatemia. In the specific case of secondary hyperparathyroidism (renal osteodystrophy), proper phosphate concentration would also benefit the patient by leading to an increase in endogenous calcitriol production and/or a lowering of PTH level. Therefore the early and adequate inhibition of PHEX activity could mitigate the serious consequences of renal osteodystrophy, giving patients an opportunity for an improved quality of life without the pain and mobility problems of advanced renal osteodystrophy. Hyperphosphatemia is defined in adults as an elevation of serum phosphorus above 1.67 mmol/L (5 mg/dL). Hyperphosphatemia is a common finding with many causes (Harrison""s 14Th Ed CD-ROM, McGraw Hill Health Professions Division, New York N.Y., chapter 356).
Hyperparathyroidism or renal osteodystrophy results from the progressive nature of chronic renal failure. The leading causes of chronic renal failure are diabetes (43%), hypertension (35%) and glomerulonephritis (14%) among US Medicare patients (patients over 65 years of age). (Harrison""s 14Th Ed CD-ROM, McGraw Hill Health Professions Division, New York N.Y., chapter 271, FIG. 271.1).
Hyperphosphatemia is potentially dangerous because of metastatic calcification. Although only an approximate guide, a calcium-phosphorus product [serum Ca (mg/dL) X serum P (mg/dL)] greater than 70 indicates a potential threat of calcification. Patients with this disease suffer from bone and joint pain, osteopenia, deformities, fractures, muscle weakness and extra-skeletal calcification.
Irrespective of the underlying cause, the disease is characterized by a progressive loss of the kidney ability to eliminate waste, to produce calcitriol (1,25(OH)2D3) and to excrete phosphate. Increased phosphate excretion is achieved with elevated PTH.
The direct effect of phosphate on PTH levels is well documented. In the presence of increasing phosphate concentration, intact fresh parathyroid gland showed increased PTH secretion (Almaden, 1996). A high phosphate diet causes elevated PTH while maintaining normal serum phosphate; in contrast, parathyroidectomized rats fed the same diet showed elevated phosphate levels (Borle, 1981 and Demeter 1991). Results in patients with mild to moderate renal failure showed that phosphate concentration correlated directly with PTH (Kates, 1997).
Although the treatment of disorders involving an inappropriate expression of PHEX is a primary goal of the present invention, the opposite is under the scope thereof. Compositions comprising a soluble active PHEX or a nucleic acid encoding same for the treatment of disorders involving PHEX deficiencies is an object of this invention.
The zinc metallopeptidase family (also known as the Zincins; see Hooper FEBS Letters 354, 1-6, 1994) is characterized by the presence of a zinc atom at the active site. This large family consists of several sub-classes that can be distinguished by their active site structure. One such sub-family is the gluzincins, which is characterized by the HEXXH motif and a glutamic acid as the third zinc ligand. This sub-family includes thermolysin, ACE (angiotensin converting enzyme), aminopeptidases and enzymes of the Neutral Endopeptidase or Neprilysin (NEP) family. NEP itself is now considered the prototype for the enzymes of the family (Crine 1997). These peptidases share extensive sequence and structural similarities. In addition to NEP, there are five other NEP-like enzymes in the public domain: the endothelin-converting enzymes ECE-1, ECE-2, Kell, XCE and PHEX (for a review, see: Turner and Tanzawa, 1997b). Several family members can cleave the same peptide substrates and the same inhibitor can inhibit more than one NEP-like enzyme. In fact, several chemical entities are capable of inhibition of more than one enzyme of the gluzincin sub-family (Roques B. P. Path Biol 1998 46,3,191-200). Therefore, known gluzincins inhibitors can be assayed in a PHEX enzymatic assay and identified as a PHEX inhibitor. Among the methods of this invention is the administration of xe2x80x9cPHEX inhibitorsxe2x80x9d. As referred to herein, the term xe2x80x9cPHEX inhibitorxe2x80x9d includes any compound that inhibits the enzymatic action of PHEX.
Towards this objective, we have prepared various reagents and tools designed to produce recombinant forms of PHEX and to purify both the recombinant and native enzymes from cell fractions, spent culture media and tissue extracts. We have cloned a CDNA encoding the full-length human PHEX protein into various expression vectors. These PHEX-encoding vectors were introduced by transfection into various cell lines including COS-1 (monkey kidney) cells, CHO (Chinese Hamster Ovary) cells, and LLC-PK1 (porcine kidney) cells. Permanent cell lines were established and shown to stably express the PHEX protein at the cell surface. A procedure was established to rapidly prepare a membrane fraction enriched in the recombinant PHEX protein.
PHEX is an intrinsic membrane protein anchored by a hydrophobic 20 amino acid sequence located near the N-terminus. The purification of an intrinsic membrane-bound protein requires the use of detergents to free it from the lipidic environment of the membrane. These detergents can interfere with the catalytic activity of the enzyme. Moreover, the detergent-purified proteins usually present stability and solubility problems, especially if concentrated solutions and/or large amounts of the protein are needed, such as those required for crystallization and high throughput screening assays. To facilitate the preparation and purification in high yields of a fully active enzyme, it is thus preferable to work with a soluble form of PHEX. Soluble forms of NEP (Lemay et al., 1989) and ECE (Korth et al., 1997) consisting of the entire ectodomain but lacking the cytosolic and hydrophobic transmembrane domains have been constructed and shown to possess enzymatic activities identical to those of the native membrane-bound homolog. A soluble form of recombinant PHEX was thus constructed by modification of the signal peptide/transmembrane region of the protein. The soluble PHEX comprises PHEX ectodomain or a catalytic part thereof; this soluble form of PHEX is referred to as secPHEX. The expression vector encoding secPHEX was transfected into LLC-PK1 cells and a permanent cell line expressing the chimeric PHEX protein on a stable basis was established. Analysis of the spent medium of this cell line by Western blot was shown to contain high levels of a secPHEX. This secPHEX was purified using either immunoaffinity or ion exchange chromatography. Ion-exchange chromatography was found to be the most efficient method to purify secPHEX from spent culture medium. The purified secPHEX was shown to be active in an enzymatic assay using PTHrP107-139 as a substrate. Moreover, the availability of this secPHEX rendered possible its use as an antigen for the production of anti-PHEX antibodies.
Monoclonal antibodies specific for PHEX were generated by immunizing mice with a PHEX-derived recombinant fusion protein produced in E. coli. These monoclonal antibodies were used to purify recombinant PHEX by various immunoaffinity procedures. PHEX-specific monoclonal antibodies also proved useful for characterizing PHEX expression in bone by immunohistochemical techniques and Western blotting.
The present invention also relates to compositions for treating PHEX-related disorders in humans and animals. The present invention particularly provides compositions for the treatment of hyperphosphatemia, including its most frequent manifestations, secondary hyperparathyroidism and renal osteodystrophy. The compositions comprise an anti-PHEX molecule which, by inhibiting PHEX activity, induce an increase in phosphate excretion as well as a reduction in gut phosphate absorption, thus reducing and/or preferably preventing hyperphosphatemia and the appearance of its most frequent consequences, secondary hyperparathyroidism and renal osteodystrophy. For example, with such a treatment, normophosphatemia is maintained in patients with mild kidney failure at the expense of PHEX activity as opposed to an increase in PTH serum concentration. While the phenotype resulting from PHEX mutation suggests that PHEX inhibition may be toxic, an attentive study of the physiology suggests otherwise. The dominant nature of the phosphate excretion suggests that only partial inhibition may be required to achieve the desired result. Observations in heterozygous females indicate that an inhibition of much less than 50% is required. At this low level of inhibition, the other features of XLH that are gene-dosage-dependent or phosphate-dependent may not be significant.
Accordingly, a first object of this invention is to provide compositions comprising PHEX enzyme or mutants, or anti-PHEX ligands. These compositions are particularly useful for treating PHEX-related disorders in humans and animals. Further objects include the provision of the following: (1) diagnostic kits for detecting the presence or amount of PHEX in a sample; (2) a method for detecting the presence or amount of PHEX in a sample; (3) devices for purifying PHEX or mutants thereof; (4) devices for screening PHEX ligands; (5) a method for obtaining a PHEX ligand; and (6) enzymatic assays involving PHEX and a PHEX substrate.